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ORIGINAL ARTICLE
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Utility of morphometry in breast carcinoma and its correlation with grade and MIB-1 labelling index


1 Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India
2 Department of Community Medicine, Armed Forces Medical College, Pune, Maharashtra, India

Date of Submission30-Mar-2021
Date of Decision18-Sep-2021
Date of Acceptance18-Sep-2021

Correspondence Address:
Puneet Baveja,
Armed Forces Medical College, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_234_21

  Abstract 


Introduction: Breast cancer is the leading cancer in women worldwide. In breast cancer, measurement of nuclear proliferation can be used in conjunction with other tumor parameters such as grade, lymphovascular invasion, and nodal status for prognosis. Various techniques have been developed to study the proliferation rate using immunohistochemistry (IHC) with Ki67/MIB-1. Employing morphometry has been reported to add accuracy to proliferation counts. The present study was done with an aim to study the pattern of MIB-1 labeling index by morphometry. Methodology: This is a descriptive study carried out at a tertiary care center in Western Maharashtra over a period of 18 months wherein 94 cases were studied. Maximum infiltrating tumor was assessed on H and E-stained sections. MIB-1% was determined using IHC and nuclear size was determined using morphometry. Results: There was a weak correlation between MIB-1 index (%) of Grade 1 tumors and their average nuclear sizes which were not significant (P = 0.509) whereas the correlation between MIB-1 index of Grade 2 and 3 tumors and their average nuclear sizes had a strong correlation with P < 0.05. Overall, it was seen that MIB-1 (%) index and nuclear sizes had a strong correlation with P < 0.05. Conclusions: MIB-1 labeling index significantly correlated with the average nuclear size of the tumor and MIB-1 labeling index can serve as a useful adjunct to histological grading of the tumors. In patients with a low histological Grade but high MIB-1 labeling index suggesting a poorer prognosis, caution may be exercised with an aggressive line of management.

Keywords: Breast carcinoma, morphometry, MIB-1 labeling index



How to cite this URL:
Verma N, Baveja P, Mahajan S. Utility of morphometry in breast carcinoma and its correlation with grade and MIB-1 labelling index. Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2022 Dec 7]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=331495




  Introduction Top


Breast cancer is the leading cancer in women worldwide.[1] Age-adjusted incidence rate in India is 25.8/100,000 women. It is second most common cause of carcinoma death in women, with more than 1.7 million cases occurring worldwide annually.[2] In breast cancer, measurement of nuclear proliferation can be used in conjunction with other tumor parameters such as grade, lymphovascular invasion, and nodal status as a prognostic indicator. Information on proliferation rate is also necessary for the development of therapeutic agents, some of which may be targeted directly at specific points in the cell division pathway.[3]

Various techniques have been developed to evaluate and quantify the proliferation rate in laboratory. Mitotic count estimates, detection of cells undergoing DNA synthesis using flow cytometer, and expression of nuclear antigens using immunohistochemical techniques have been used. Immunohistochemistry (IHC) methods are cheaper, quicker, and easier to use than flow cytometry and autoradiography. Furthermore, they are more reliable and reproducible than mitotic figure counting. They also have the advantage of being applicable to cytological material from breast aspirates and need less tissue than some other methods.[4] Employing morphometry to assess the proliferation index has been reported to add accuracy to proliferation counts.[4]

Ki67 is a protein expressed in the nucleus during the cell cycle. Cells express this antigen during G1, S, G2, and M phases but not during G0 phase. Ki67 antibody requires only fresh or frozen tumor specimens. MIB-1 is a monoclonal antibody against the recombinant parts of Ki67 antigen. MIB-1 antibody has the advantage of being effective in fixed, archival specimens following microwave irradiation. Staining with MIB-1 clonal antibody has been shown to correlate well with histological grade, mitotic index, and overall survival.[4]

The present study was done with an aim to study the pattern of MIB-1 labeling index by morphometry as well as light microscopy in breast carcinoma cases and to find the mean MIB-1 labeling index.


  Methodology Top


This is a descriptive study carried out at a tertiary care center in Western Maharashtra over a period of 18 months. The ethical clearance was obtained by Medical Research Cell and Institutional Ethical Committee, Armed Forces Medical College, Pune – 40 vide their letter number IEC/OCT/2018 dated 22 Oct 2018.

A total of 94 cases of breast carcinoma from which representative blocks with maximum infiltrating tumor were studied retrospectively from the archives 2017 onward and prospectively 2018 onward and the following procedure was followed:

  1. H and E-stained sections of all consecutive cases including retrospective cases of breast carcinoma meeting the inclusion criteria were reviewed for the accuracy of diagnosis
  2. Block with maximum infiltrating tumor was selected for IHC
  3. MIB-1 immunostaining was scored in the highest proliferating tumor foci using a light microscope (×40 objective)
  4. IHC staining of the primary tumor was assessed for MIB-1 labeling. The percentage of positively stained cells was determined in each of 10 high-power fields spread randomly through the section and an average score was obtained, this was the labeling index for a case
  5. Mean of labeling indices was calculated
  6. Malignant cells with crisp nuclear staining of any intensity were regarded as positive
  7. For each specimen, 10 high-power fields were captured using digital camera attached with the microscope to determine nuclear size. To perform the same, the morphometry software “Dewinter Biowizard” was used as shown in [Figure 1]
  8. At least 1000 cells were counted in all samples.[5]
Figure 1: Morphometric results using digital imaging Biowizard 4.1 Software

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Inclusion criteria

All cases of breast carcinoma with available archival or surgical specimen tissue.

Exclusion criteria

Postradiotherapy and chemotherapy cases of breast carcinoma, breast carcinoma cases with inadequate tissue for the assessment, and metastatic and nonepithelial malignancies of breast.

The sample size was calculated based on the study conducted by Nakagomi et al.[5] At 95% Confidence Interval with a standard deviation (SD) of 12 and absolute error of margin as 2.5 sample size came out to be 89. However, 94 cases were available for the study.

Descriptive statistical analysis was used for the results wherein results on continuous measurements are presented as mean ± SD and results on categorical measurements are presented in number (%). Chi-square test was used for the comparison of mean MIB-1 labeling index scores in different grades and invasion status. Two-tailed tests were used and P < 0.05 was considered statistically significant. Data analysis was done using the SPSS software version 23.0.


  Results Top


The mean age of patients whose sections were studied was 54.8 ± 10.92 years (minimum 30 years and maximum 88 years). The age distribution of the patients is given as per [Table 1].
Table 1: Age distribution

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Of the 94 patient samples, 3 (3.19%, 95% confidence interval [CI] 0.66%–9.04%) were Grade 1 tumors, 51 (54.26%, 95% CI 43.66%–64.58%) were Grade 2 tumors, and 40 (42.55%, 95% CI 32.41%–53.18%) were Grade 3 tumors. It was noted that in Grade 1 tumors mean of MIB-1 (%) was 6% with a range of 5%–8%. In Grade 2 tumors, mean of MIB-1 (%) was 9.33% with a range of 2%–32%. In Grade 3 tumors, mean of MIB-1 (%) was 20.9% with a range of 3%–55%. The grading of tumor was evaluated in the context of average nuclear size and MIB-1 (%). Descriptive statistics of average nuclear size as per the grade of tumor is as per [Table 2].
Table 2: Descriptive statistics of average nuclear size as per the grade of tumor

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Pearson's correlation was used to find the association between MIB-1 (%) and average nuclear size as per the grade as depicted in [Table 3]. It was seen that there was a weak correlation between MIB-1 index (%) of Grade 1 tumors and their average nuclear sizes which were not significant (P = 0.509) whereas the correlation between MIB-1 index of Grade 2 and 3 tumors and their average nuclear sizes had a strong correlation with P < 0.05. Overall, it was seen that MIB-1 (%) index and nuclear sizes had a strong correlation with P < 0.05.
Table 3: Association between MIB - 1(%) and average nuclear size as per the grade by Pearson's Correlation (2 Tailed)

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  Discussion Top


In our study, the mean age was found to be 54 ± 10.92 years (minimum 30 years, maximum 88 years) which was in concurrence with other studies. In a study by Chopra et al.[6] authors studied the trends of breast carcinoma with regard to age. They evaluated 100 breast carcinoma specimens and noted that two distinct peaks of breast cancer occurred between 41–50 and 51–60 years of age. The mean age of the patients in their study was 50.1 years (SD ± 10.5) which was similar to our study where mean age was 54.8 years.

In our study of the 94 patient samples, 3 (3.19%) were Grade 1 tumors, 51 (54.26%) were Grade 2 tumors, and 40 (42.55%) were Grade 3 tumors. In a study by Laishram et al.[7] in a sample size of 142 cases, authors noted that the majority of the cases were Grade 3 at 57.04% (n = 81 cases). They observed a high percentage of high grades and late presentations of breast carcinoma in the study and attributed it to lack of proper screening and awareness programs. This typically represents the Indian scenario and this gets amplified in rural setups. Similar findings were seen in our study where most of the cases were seen in Grade 2 and 3 breast carcinomas. Study by Contesso et al.[8] in a sample size of 1010 patients showed that they had 50% of Grade 2 patients.

Our study showed positive association between the grade of the tumor and its average nuclear size. This finding of our study was supported by a study conducted by Tambasco and Magliocco[9] on 97 patients wherein they had 31% patients in Grade 1, 42% patients in Grade 2, and 27% in Grade 3. They found mean fractal dimension (includes nuclear pleomorphism and mitosis) increases with tumor grade which was statistically significant. Spyratos et al.[10] and many other authors in their studies observed significant relation between MIB-1 labeling index and tumor grade. These authors also noted nuclear pleomorphism correlated with MIB-1 labeling index. Similar finding was brought out by Kashyap et al.,[11] where they performed nuclear morphometry on 720 breast carcinoma cases. They also concluded that nuclear morphometry correlates with tumor size, lymph node involvement, and mitotic activity.

Limitations of the study

There were a few limitations in my study like the patients were not evaluated in the context of risk factors such as menopausal age, family history, and exposure to radiation. The patient follow-up was not performed in this descriptive study. Posttreatment (postchemotherapy and postradiotherapy) patients were not included in the study. The study was done based on the average nuclear area grading; however, various histological types of tumors were not evaluated separately. Status of ER, PR, and HER-2 neu receptor status was out of the scope of the study.


  Conclusions Top


Based on our study findings, MIB-1 labeling index significantly correlated with the average nuclear size of the tumor and morphometry along with MIB-1 labeling index can serve as a useful adjunct to histological grading of the tumors. In patients with a low histological grade but high MIB-1 labeling index suggesting a poorer prognosis, caution may be exercised with an aggressive line of management.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10-29.  Back to cited text no. 1
    
2.
Laura C. Collins. Breast. In Rosai and Ackerman's surgical pathology. John R Goldblum, Laura W Lamps, Jesse K McKenney. Jeffrey L Myers editors. Elsevier, Philadelphia 2018: p.1464-5.  Back to cited text no. 2
    
3.
Beresford MJ, Wilson GD, Makris A. Measuring proliferation in breast cancer: Practicalities and applications. Breast Cancer Res 2006;8:216.  Back to cited text no. 3
    
4.
Jansen RL, Hupperets PS, Arends JW, Joosten-Achjanie SR, Volovics A, Schouten HC, et al. MIB-1 labelling index is an independent prognostic marker in primary breast cancer. Br J Cancer 1998;78:460-5.  Back to cited text no. 4
    
5.
Nakagomi H, Miyake T, Hada M, Hagiwara J, Furuya K, Muto S, et al. Prognostic and therapeutic implications of the MIB-1 labeling index in breast cancer. Breast Cancer 1998;5:255-9.  Back to cited text no. 5
    
6.
Chopra B, Kaur V, Singh K, Verma M, Singh S, Singh A, et al. Age shift: Breast cancer is occurring in younger age groups-is it true? Clin Cancer Investig J 2014;3:526.  Back to cited text no. 6
  [Full text]  
7.
Laishram RS, Jongkey G, Laishram S, Sharma D. Clinico-morphological patterns of breast cancer in Manipur, India. Int J Pathol 2011;9:40-3.  Back to cited text no. 7
    
8.
Contesso G, Mouriesse H, Friedman S, Genin J, Sarrazin D, Rouesse J. The importance of histologic grade in long-term prognosis of breast cancer: A study of 1,010 patients, uniformly treated at the Institut Gustave-Roussy. J Clin Oncol 1987;5:1378-86.  Back to cited text no. 8
    
9.
Tambasco M, Magliocco AM. Relationship between tumor grade and computed architectural complexity in breast cancer specimens. Hum Pathol 2008;39:740-6.  Back to cited text no. 9
    
10.
Spyratos F, Ferrero-Poüs M, Trassard M, Hacène K, Phillips E, Tubiana-Hulin M, et al. Correlation between MIB-1 and other proliferation markers: Clinical implications of the MIB-1 cutoff value. Cancer 2002;94:2151-9.  Back to cited text no. 10
    
11.
Kashyap A, Jain M, Shukla S, Andley M. Role of nuclear morphometry in breast cancer and its correlation with cytomorphological grading of breast cancer: A study of 64 cases. J Cytol 2018;35:41-5.  Back to cited text no. 11
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    Figures

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    Tables

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