Introduction
Micronuclei scoring can be used as a bio-marker of genotoxic and chromosomal damage. Micronucleus (MN) is a small fragment of nucleus that does not get included into the daughter cell during the process of cell division. Due to chromosomal aberrations sometimes there is a lag in whole chromosomes during cell division.1 The Micronuclei are seen as round structures and are similar in texture and colour to the main nucleus and are are 1/3 to 1/16th the size of the main nucleus when visualized under oil immersion. They are seen in the cytoplasm of the cell.2 Fine needle aspiration cytology (FNAC) is applied as the primary tool for diagnosis in breast masses because of its ease and rapidity.3, 4, 5 Borderline lesions are difficult to ascertain on FNAC and in such situations, Micronuclei become important as they provide an objective and reproducible diagnosis.6, 7, 8 Micronucleus (MN) scoring can be carried out in benign (fibroadenoma) and malignant (infiltrating ductal carcinoma) breast lesions to evaluate the role of Micronuclei as a biomarker in breast carcinomas.9 Often the “Triple test” ie, the clinical examination, mammograpic impression and fine needle aspiration cytology (FNAC) are used in conjunction for diagnosis of breast masses.10 To this, we can further add the Micronuclei scoring to refine the diagnostic approach.
Materials and Methods
Ethical institutional permission was taken. This was a prospective study done in the Department of Pathology at A.C.S Medical College and Hospital for a duration of two years from June 2019 to May 2021. A total of 60 breast cytology smears were examined for micronuclei.
Criteria for micronuclei
Diameter of Micronuclei should be 1/16 to 1/3 the diameter of the main nucleus.
The shape, colour and texture of Micronuclei should be similar to those of nucleus.
Staining intensity should be similar to, or slightly weaker than that of the nucleus.
Micronuclei should be round to oval in shape having close proximity but no actual contact with the nucleus.
Plane of focus should be same as that of the main nucleus.
Methodology
A total of 60 breast lumps and diffuse swelling cases were sent for FNAC to the department of Pathology from the department of General Surgery. FNAC was done in all the cases under aseptic conditions. The slides were stained with Hematoxylin and Eosin and also Papanicolaou stain. All the slides of breast lesions were screened and diagnosis was reported.
In addition, Micronuclei scoring was done on 1,000 cells on H &E stained smears under oil immersion (×1,000). Scoring was done according to Fenech et al.11 The zigzag method was followed for screening of slides. 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 and cells that had indistinct nuclear morphology were not considered for micronuclei scoring.
Two pathologists observed the slides independently and a mean value was taken. The average score was compared between the benign, adenosis, usual hyperplasia, atypical hyperplasia, and carcinomas. Cases of malignancy were again graded into grade I, grade II and grade III based on grading system by Robinson et al.12
Data analysis
Data was entered in Microsoft Excel sheet and analyzed using SPSS version 20.0 statistical software. Data was depicted in the form of tables, graphs, percentage and proportion. Mean, standard deviation of continuous variables, sensitivity, and specificity, Mann Whitney test, unpaired t test with Welch's correction was used and Area under the receiver operating curve (ROC) was also used. The value of P < 0.05 was taken as statistically significant.
Results and Observation
Table 1
Age distribution
|
Age distribution (years) |
No. of cases |
Percentage (%) |
|
20-30 |
30 |
50% |
|
31-40 |
17 |
28.3% |
|
41-50 |
08 |
13.3% |
|
51-60 |
02 |
3.3% |
|
>60 |
03 |
5% |
|
Total |
60 |
100% |
In the present study, majority of the cases were noted in 20-30 years constituting 50% cases.Table 1
Clinical presentation: Well defined breast lump was noted in 88.3% (53/60) cases and 11.6 % (07/60) cases showed diffuse swelling.
Laterality of lesions: On site of presentation, 43.3% cases presented with swelling on right side, 36% (22/60) on left side and 20% (12/60) presented had both sides lesion.
Table 2
Distribution of Breast lesions on Cytology
|
Breast lesion |
No. of cases |
Percentage (%) |
|
Fibroadenoma |
38 |
63.3% |
|
Adenosis |
10 |
16.6% |
|
Usual ductal hyperplasia |
04 |
6.6% |
|
Atypical ductal hyperplasia |
02 |
3.3% |
|
IDC |
06 |
10% |
|
Total |
60 |
100% |
The total number of breast cytology aspirate patients was 60. Fibroadenoma was the most common reported diagnosis and was seen in 63.3% cases.Table 2
Figure 1
(A-F) Arrows point the micronucleiin the cytoplasm of 4 cases of infiltrating ductal carcinoma of the breast. (A,C,E: H&E stain, x 400; B,D,F: H&E stain, x 1000)
Table 3
Average score range of micronuclei
Fibroadenoma: 38 cases were reported as fibroadenoma on FNAC. The average age group of women with fibroadenoma was 28.5 ± 4.72 years. The mean micronucleus score of the benign cases was 1.42 ± 0.85 (Range 0–4). MN score of ≤1 had a high sensitivity (100%) and specificity (99%) in confirmation of benign cases.
Adenosis: Among 10 cases of adenosis, 3 cases were sclerosing adenosis, 1 case was micro glandular adenosis, and 6 cases were fibroadenosis. The mean age of this group was 37.3 ± 3.9 years. The average micronucleus score (per 1000 epithelial cells) of this group was 2 ± 0.
This micronucleus score was very mildly higher than the fibroadenoma group but statistically significant (P <<0.0001) (Mann Whitney test)
Table 4
Mann whitney test for fibroadenoma, UDH and ADH
Usual ductal hyperplasia (UDH) constituted 04 cases. The mean age of this group was 40.25 ± 4.6 years. The average micronucleus score (/1000 epithelial cells) was 4 ± 0 higher than the benign and adenosis group and statistically significant (P < 0.0001) (Mann Whitney test) Atypical ductal hyperplasia (ADH) constituted 2 cases with an average age of 45.5 ± 3.5 years. The mean micronucleus score (/1000 epithelial cells) of this group was 6 ± 0.0. The difference in the micronucleus score was statistically significant (P < 0.0001). (Mann Whitney test) (Table 4)
Infiltrating ductal carcinomas 06 cases showed Micronucleus score of ≥5 and with an average age of 58.8 ± 8.2 years. Unpaired t test (with Welch’s correction) showed significant difference in Micronuclei score between malignant and benign breast lesions (P = 0.0046, df = 7.018) (Table 5). There was increase in micronucleus scores from the benign to the malignant lesions.
Table 5
Unpaired t test with welch's correction
|
Unpaired t test with Welch's correction |
|||
|
P value |
0.0046 |
||
|
P value summary |
** |
||
|
Significantly different (P < 0.05)? |
Yes |
||
|
One- or two-tailed P value? |
Two-tailed |
||
|
Welch-corrected t |
df |
t=4.088 |
df=7.018 |
ROC curve – micronuclei score for lesion differentiation. Area under the curve is 1. Micronucleus score > 4 had a sensitivity and specificity of >97 %.
Area under the ROC curve: This was taken as 1. The Std error was 0. The 95% confidence interval was 1.000 to 1.000 and the P value was <0.0001.
Robinsons grading of IDC: Robinsons grading was done on cytology smears. 02 cases were grade 1 with average micronucleus score /1000 epithelial cells was 8, 02 cases were grade 2 and the average micronucleus score was 18.5 and 02 cases were grade 3 with average micronucleus score was 27.5. The range of micronucleus for Grade 1, 2 and 3 was 5-25, 15-30 and 20-30 respectively.
Discussion
Accumulation of damaged genetic material is commonly seen in most of the cancers. Micronuclei are bits and parts of chromosomes or even whole chromosomes that do not get included in the daughter nuclei during division. In the present study, we have attempted to look at these micronuclei on breast aspirate smears and ascertain their features in benign and malignant conditions of breast.
Comparative studies on cytological diagnoses distribution
In the present study, the total number of breast cytology aspirates for a period of two years in the department of Pathology was 60 cases. Out of these 60 cases, 38 cases were reported as fibroadenoma, 10 cases as adenosis, 04 cases as usual ductal hyperplasia, 02 cases as atypical ductal hyperplasia, 6 cases of infiltrating ductal carcinomas. In a study conducted by Sylvia MT et al13 there were 190 cases of benign tumours (fibroadenoma/phyllodes); 90 cases of benign proliferative breast disease, 30 cases of cystic diseases (fibrocystic disease, abscess, simple cysts), 7 cases of adenosis, 11 cases of usual ductal hyperplasia, 5 cases of atypical ductal hyperplasia and 30 cases of infiltrating ductal carcinomas. Basher ES et al14 examined (74) benign breast tumors and (26) malignant tumors (Infiltrating ductal carcinoma, not otherwise specified).
Comparative studies related to age distribution
In our study the average age women with fibroadenoma was 28.5 ± 4.72 years and that of IDC was 58.8 ± 8.2 years. In Basher ES et al14 study, there were 74 benign breast tumors and 26 malignant tumors (Infiltrating ductal carcinoma, no otherwise specified). All patients were females. The peak incidence of benign tumors was in the third decade of life while malignancy was more frequent in the fifth decade. In Sylvia MT et al study13 the average age group of women with fibroadenoma was 25 years and phyllodes was 60 years. The mean age of this group was 25 years.In Mangam SN et al study 15 the mean ages (± standard deviation) of the fibroadenoma and IDC groups were 28.74 ± 8.44 and 49.84 ± 12.62 years, respectively.
Micronucleus scores in benign lesions and Invasive ductal carcinoma (IDC)
In the present study, the mean micronucleus score of the benign cases was 1.42 ± 0.85 (Range 0–4). In a study conducted by Sylvia MT et al13 the mean micronucleus score of the benign cases was 0.5 ± 0.52 (Range 0–1).
There was an increase in micronucleus scores from the benign to the malignant lesions. An Unpaired t test (with Welch’s correction) showed significant difference in Micronuclei score between malignant and benign breast lesions (P = 0.0046, df = 7.018). Similar findings were observed Sylvia MT et al. 13
Hemalatha et al16 in their study reported a significant difference in occurrence of micronuclei between benign and malignant cases as well as between various grades of malignancy. There was overlapping of values between few fibroadenoma cases and infiltrating ductal carcinoma and a score of less than 5 for 1000 cells was seen in most cases of fibroadenoma, and a higher score of more than 5 per 1000 cells was seen in infiltrating ductal carcinoma. Independent Samples Test (Student t test) showed significant difference between benign and malignant groups (P < 0.000). In Mangam SN et al15 study, comparisons of mean MN scores of fibroadenoma with cytological grades in the Giemsa-stained smears showed that the mean MN scores (± standard deviation) of fibroadenoma and Grades I, II and III IDC were 0.13 ± 0.34, 3.80 ± 1.57, 9.34 ± 3.93 and 15.51 ± 5.90, respectively. The MN score increased significantly in a stepwise manner from Grades I to II, II to III of IDC. Basher ES et al14 observed Micronucleus score of ≥10 had a high sensitivity (96%) and specificity (99%) of detecting malignancy. The area under the receiver characteristic operating curve (ROC) for this category was very significant 0.995. Goel S et al 17 studied spontaneously occurring MN and counted them in epithelial cells on fine needle aspiration cytology (FNAC) smears in 50 patients with benign and malignant breast lesions. All these were then correlated with grades of breast cancer at cytology. Most IDC cases showed variable number of MN (n = 16, MN mean = 9.3), in contrast to the benign lesions (n = 26) where they were consistently absent.
Comparison based on robinsons grading system
In our study, we observed 02 cases were grade 1 with average micronucleus score /1000 epithelial cells of 8, 02 cases were grade 2 and the average micronucleus score was 18.5 and 02 cases were grade 3 with average micronucleus score of 27.5. In Sylvia MT et al study 13 10 cases (33.3%) were grade one, 14 cases (46.6%) were grade two, and 6 cases (20%) were grade three tumors, and the average micronucleus score of these groups were 13.2 ± 5.7, 20.36 ± 8.5, and 27.5 ± 4.18, respectively.
Preoperative pathology diagnosis is an important part of the work up for breast lesions. Often biopsy of the breast lump is recommended whenever there is a strong clinical suspicion of malignancy.18 On regular FNAC reporting, not much attention is paid to the micronuclei. The micronuclei scoring on routine FNAC aspirates of IDC is relatively easy, reliable and reproducible.19 Hence, features of micronuclei serve as one of the indicators to delineate the benign from malignant lesions in breast aspirates.
