Establishment of Correlation of Ki-67 Proliferative Index Expression in the Peritumoral Tissue with Tumor Mass in ER (Estrogen Receptor) Positive Breast Carcinoma

Background: Breast carcinoma is the second most frequently occurring malignant tumor. It usually arises in a multistep fashion from intermediary lesions to invasive cancer. Identifying such predominantly occurring lesions adjacent to malignancy and studying of Proliferative Ki-67 index and (ER) status in such lesions and substantiate their possible identity as a premalignant lesion. The study tries to establish the lesions which have the potential for progression to overt malignancy, thereby indicating early identification and appropriate treatment. Aim: To establish a correlation in the expression of Ki-67 proliferative index in the peritumoral tissue with tumor mass of ER-positive breast carcinomas. Objectives: 1.To study the expression of ER in breast tumor mass. 2. To study expression of Ki-67 proliferative index in breast tumor mass and peritumoral tissue. 3. To assess the expression of Ki67 proliferative index in breast tumor mass and peritumoral tissue. Study design: Observational prospective study. Original Research Article Sukanya et al.; JPRI, 33(61B): 24-33, 2021; Article no.JPRI.80634 25 Materials and Methods: 32 mastectomy specimens of diagnosed cases of breast carcinoma sent to the Histopathology Section are studied. Gross features are described and the histopathological section stained by (H & E) stain and immunohistochemistry using monoclonal antibody to (ER) and proliferation-associated antigen (Ki-67) are studied. Statistics: Chi-square test, Spearman’s rankorder correlation coefficient, and software used in the analysis were SPSS 22.0 version and Graph Pad Prism 6.0 version and p<0.05 is considered as the level of significance. Results: In this study out of 32 ER-positive cases, 24 showed positivity for Ki-67 in tumor mass and 22 cases showed high Ki-67 in peritumoral tissue. Maximum lesions were proliferative lesions with atypia like ADH and DCIS. Conclusion: Ki-67 is an accurate nuclear proliferative marker to assess cell proliferation status. In breast carcinoma adjacent peritumoral lesions show a high proliferative index which suggests a link and a trail of generation of premalignant lesions converting to invasive carcinoma. Proliferative lesions with and without atypia, moderate epithelioid, atypical ductal hyperplasia, and DCIS more or else equals IDC for Ki-67 proliferative index. Such cases should be regularly followed up for progression of these lesions to malignancy.


INTRODUCTION
In women following cervical carcinoma, breast carcinoma stands as the most commonly occurring solid epithelial tumor. Nowadays it is not a rarely diagnosed condition causing deaths. Worldwide in developed and developing countries, it is a leading cause of death in the female population [1]. Breast carcinomas are observed to progress in a multistep fashion, ranging from benign to lesions with intermediate potential, to finally invasive carcinoma [2,3]. These before malignant lesions should thus be identified at an early stage to prevent progression to malignancy. Breast lesions are described as proliferative and neoplastic. Estrogen is a hormone responsible for proliferative activity and progression of the tumor. Estrogen Receptor (ER) status and proliferative index (Ki-67) are important markers of disease progression and prognosis [4]. Hence they are useful prognostic factors predicting survival free from disease in these patients [5,6].
Different lesions are seen in the same breast, the area in proximity to malignancy, justifying the multistep progression to malignancy [1,2]. Benign breast disease (BBD) is a group of lesions like atypical ductal hyperplasia, atypical lobular hyperplasia, ductal and lobular carcinoma in situ seen on histopathology. These have further been categorized into three broad pathologic categories: non-proliferative, proliferative without atypia, and proliferative with atypia [7]. Benign proliferative breast disease is an extremely complex and interrelated group of proliferative disorders of the breast parenchyma. They do not necessarily mean neoplastic lesions, rather they are a result of hormone-induced hyperplastic processes. Thus studying such lesions adjacent to malignancy and knowing Ki-67 proliferative index (MIB-1 index) and Estrogen receptor (ER) hormonal status in these cases would help us identity premalignant lesions in the respective patient.
The proportionate correlation between ER positivity and the Ki-67 index has been established in this study by learning their expression. The study identifies the lesions possessing potential for progression to frank malignancy. Thus early identification shall help to target therapy and ultimately reduce the incidence of breast cancers.

METHODS AND MATERIALS
The study was done from 1 st August 2017 to 31 st July 2019 in the histopathology division of the Department of Pathology in association with the Surgery department, JNMC Sawangi (Meghe), Wardha, Maharashtra. It is an observational prospective study.

Sample Size [8]
The sample size came out as 30, derived from the formula, n=Zα/2 2 . P.
where "Z a/2" is the level of significance at 5 % that is 95 % confidence interval. "p" is the prevalence of oral squamous cell carcinoma.
"d" is the desired error of margin. "n" is the sample size.

METHODOLOGY
Consent in a well-informed manner was taken from all the patients who came with complaints of a breast lump. Clinical assessment and proper history were obtained from these patients who were inpatients of the surgery department. FNA or true-cut biopsy was performed in most of the cases and diagnosed breast carcinoma patients were chosen for the study. These patients were subjected to MRM. Tissue specimens obtained from MRM procedure were received in the division of Histopathology, Department of Pathology, and kept overnight for fixation.• In the next step the grossing of specimens as per the standard method given in protocol. Sections from the tumor mass and peritumoral tissue were taken.  Out of 32 ER, Ki-67 positivity in tumor mass was seen in 24 cases and in peritumoral tissue in 22 cases.

Definition of peritumoral tissue-It
The highest Ki-67 expression was seen in the category of proliferative changes with atypia and in situ changes that included cases of ADH and DCIS (Fig. 1).
Thus, proliferative lesions were more common than non-proliferative lesions. The commonest histomorphological change observed was of Moderate epitheliosis i.e. 12 cases (38%) while the least evident finding was of fibrocystic change accounting for a single case (03%).
The expression of Ki-67 was high particularly in cases of ADH and DCIS (Fig. 2).

Proliferative changes with atypia
Proliferative changes without atypia Benign nonproliferative changes

Statistics
Statistical analysis was carried out by using descriptive and inferential statistics using chisquare test, Spearman's rank-order correlation coefficient and software used in the analysis were SPSS 22.0 version and GraphPad Prism 6.0 version while p<0.05 is taken as the level of significance.
The p-value for ER positivity and Ki-67 positivity in tumor mass was found to be 0.002 which is significant. p-value drawn for the Ki-67 proliferative index in peritumoral lesions was also found to be 0.001, which was significant irrespective of the histomorphological lesions within its categories.

DISCUSSION
Shashikala et al. [10] in their study, proliferative fibrocystic lesions in association with carcinoma breast-Study of mastectomy specimens, have a maximum number of cases accounting 65%, to undergo the diagnosis and subsequent mastectomy for IDC in the age range of 41-60 years. The present study has a concordant observation regarding the age range for the diagnosis of the IDC. Niikura et al. [11] in their study too had a mean age of presentation as 51 years.
The BR scores of the studies Klintman et al. [12] and Niikura et al. [11] were similar to the BR scores of the present study where peritumoral tissue assessment of the ER-positive breast is done, maximum number of patients belonged to BR Grade 2 (Moderately Differentiated) i.e. 18 cases (57%).
The consistent peritumoral area histomorphology has also been reported in the studies of Shashikala et al. [10] and Sathyalakshmi et al. [8].
Zhou [13], studied ER and Ki-67 expression in 56 cases, that constituted all proliferative lesions with and without atypia like UDH, ADH and DCIS. for peritumoral histomorphology and Ki-67 proliferative index. A total of 09 cases showed ER positivity. Increased ER positivity was commoner in proliferative lesions with atypia including in situ lesions. Most of the proliferative lesions without atypia were negative for ER and some came with little ER positivity, which is in concordance to our study.
The similar observation for Ki-67 proliferative index for ER positive IDC for the tumor mass has been quoted in the studies of Sathyalakshmi et al.
[8] 7 cases of proliferative lesions with atypia and 8 cases of proliferative lesions without atypia showed Ki-67 positivity. The Ki 67 positivity was seen very high in patients with lesions such as ADH and DCIS. While only one case showed low Ki-67 expression and that belonged to benign non-proliferative category. These findings are in concordance with the present study. Ki-67 proliferative index for peritumor tissue in the agreement of the observation of present study has also been recorded in the studies of Zhou [11], where lesions showing ER positivity with high Ki-67 proliferative index mainly were proliferative lesions, especially ones with atypia and in situ lesions. These included following lesions-UDH, ADH, and DCIS. Similar related studies were reported [14][15][16][17]. Studies reported by Yeola et al. [18,19] Anand et al. [20] were reviewed.
The present study also made a similar observation to that of Sathyalakshmi et al. [8] where high Ki-67 proliferative index in addition to ER positivity was observed with situations of atypical ductal hyperplasia and DCIS.

LIMITATIONS
The comparative statistics for Ki-67 proliferative index in the lesions observed in the peritumoral tissue available in the literature is less. The observations made in the present study therefore could not be compared extensively. The followup studies of histomorphological alterations in the peritumoral tissue of ER positive IDC too are not in plenty in published literature. Therefore one of the limiting factor for the present study is the follow-up of such cases even though it is one time Ki-67 estimated is high.

RECOMMENDATIONS
The reporting protocol of the mastectomy specimen should include the assessment of histomorphological alterations in the peritumoral tissue with its specified category and lesions within it. ER positive IDC should undergo Ki-67 proliferative index in the tumor as well the peritumoral tissue so that the proliferating potential can be predicted for recurrence and subsequent metastasis in the case of IDC.

CONSENT
Informed consent was taken from the target patients.

ETHICAL APPROVAL
The study was proceeded with, after the approval of the Institutional Ethics Committee.

ACKNOWLEDGEMENT
The study was a funded project, supported by Indian Council of Medical Research, New Delhi, India. The authors gratefully acknowledge the dedicated efforts of the investigators, coordinators and volunteer patients recruited in the study.