Expression of Ki-67 and E-cadherin in patients with non-small cell lung cancer attending a tertiary care hospital

Background: E-cadherin and Ki-67 expressions may provide real-me insights into the tumor's status and can be u�lized as targeted therapeu�cs for lung cancer. We aimed to explore the expression of Ki-67 and E-cadherin in non-small cell lung cancer (NSCLC) pa�ents and to iden�fy their associa�on with clinicopathological features. Methods: In this cross-seconal study, forty formalin-ﬁxed paraﬃn-embedded (FFPE) NSCLC �ssue blocks were iden�ﬁed from January to October 2022, based on hospital records from the Department of Pathology of Na�onal Ins�tute of Diseases of the Chest and Hospital, Dhaka Medical College and Hospital. Samples were reevaluated for �ssue quality, diagnosis, and exclusion -inclusion criteria. Finally, twenty-ﬁve samples were analyzed, and relevant clinicopathological data were collected from pa-ents or authorized representa�ves. Ki-67 and E-cadherin expression were analyzed by immunohistochemistry and their rela-onships with each other. Results: Ki-67 expression was posi�ve in 40% of the NSCLC �ssue, and E-cadherin was nega�ve in 40% of the NSCLC �ssue. No sta�s�cally signiﬁcant rela�on was


INTRODUCTION
Lung cancer stands as a predominant cause of cancerrelated death globally.According to the cancer registry report (2018-2020), it occupied the first position (17.4%)among the cancers occurring in both sexes in Bangladesh. 1 It is thought that the failure of lung cancer treatment is primarily attributed to metastasis and recurrence, resulting in a persistently dim prognosis for this condition. 2Around 80% of total lung cancers are non-small cell lung cancer (NSCLC). 3The molecular mechanism of lung cancer is very complex.Immunohistochemistry (IHC) plays an important role in characterizing cancer cells by identifying various biomarkers.One of the molecular processes involved in lung cancer is known as epithelial-mesenchymal transition (EMT), a complex reprogramming process of epithelial cells, which plays an essential role in tumor invasion and metastasis.Loss of E-cadherin (a cell-tocell adhesion molecule) expression is one of the hallmarks of EMT. 4 E-cadherin is a transmembrane glycoprotein expressed on the cell surface and maintains cell-cell junctions, thereby inhibiting aberrant cell proliferation and migration. 5r assessment of tumor proliferation, there is a frequent reliance on the analysis of proliferationassociated antigens, particularly Ki-67, a nuclear protein. 3It is expressed throughout all cell cycle phases in actively proliferating cells, but it is not expressed in quiescent (G0) cells.As a result, Ki-67 can serve as an ideal target antigen for evaluating proliferation in NSCLC. 6Therefore, concurrent assessment of Ecadherin and Ki-67 can provide real-time insights into

ABSTRACT
Background: E-cadherin and Ki-67 expressions may provide real-me insights into the tumor's status and can be u lized as targeted therapeu cs for lung cancer.We aimed to explore the expression of Ki-67 and E-cadherin in non-small cell lung cancer (NSCLC) pa ents and to iden fy their associa on with clinicopathological features.Methods: In this cross-sec onal study, forty formalin-fixed paraffin-embedded (FFPE) NSCLC ssue blocks were iden fied from January to October 2022, based on hospital records from the Department of Pathology of Na onal Ins tute of Diseases of the Chest and Hospital, Dhaka Medical College and Hospital.Samples were reevaluated for ssue quality, diagnosis, and exclusion -inclusion criteria.Finally, twenty-five samples were analyzed, and relevant clinicopathological data were collected from paents or authorized representa ves.Ki-67 and E-cadherin expression were analyzed by immunohistochemistry and their relaonships with each other.
Results: Ki-67 expression was posi ve in 40% of the NSCLC ssue, and E-cadherin was nega ve in 40% of the NSCLC ssue.No sta s cally significant rela on was found between the expressions of Ki-67 and E-cadherin.No sta s cally significant associaon was found in Ki-67 and E-cadherin expressions with clinicopathological characteris cs except E-cadherin with comorbidity.

Conclusion:
Posi ve expression of Ki-67 and nega ve expression of E-cadherin was found in two-fi hs of NSCLC ssues but not significant.Simultaneous es mated of Ki-67 and E-cadherin may contribute to the treatment planning and predict prognosis of the NSCLC pa ents.

BRIEF ARTICLE
the tumor's status and can be utilized as targeted therapeutics for lung cancer, as proposed by He et al. 7 We aimed to explore the expression of Ki-67 and Ecadherin in NSCLC patients and to identify their association with clinicopathological features.

Validation of immunohistochemical staining
One of us (FA) scored the slides and validated them with a Histopathologist (FA).Ki-67 positive cells were identified based on brown-stained nuclei.The counting was done by identifying hot spots or by global method. 9e area with the highest number of Ki-67 positive nuclei was considered a hot spot.Positive status depends on the number of brown stained nuclei, not staining intensities. 10Four hot spots of Ki-67 positive cells were identified (if clearly seen), and counted at least 100 nuclei in each field.If the hot spots were unavailable in the slide, the global method was followed (one field with the highest Ki-67 index containing 500 nuclei).The total number of Ki-67 positive nuclei was counted and divided by the total number of counted cancer cells under high power magnification (×400).
Finally, the Ki-67 index was expressed as a percentage.
2. Negative expression of E-cadherin was found in two-fifths of NSCLC tissues.
3. A combination of E-cadherin and Ki-67 assessment can be useful for therapeutics in lung cancer.
and a final score of 0 to 12 was achieved.The final score of 0 to 3 was considered negative, and the 4 to 12 was considered an E-cadherin positive sample. 7

Statistical analysis
Statistical analysis was done using SPSS, version 25.
Data were described in terms of both number and percent.Fisher's exact test was done to assess the relationship between the expressions of E-cadherin and Ki-67.All statistical tests were two-tailed, and P<0.05 was considered statistically significant.

RESULTS
The patients of the selected samples had a mean (standard deviation) age of 56.8 (10.1) years, with 84% being male.Two-fifths had at least one comorbid medical condition, and two in every ten had a family history of lung cancer.More than half of the samples (56%) exhibited squamous cell carcinoma, and nearly half (48%) of the tumors were well-differentiated.
Lymph node metastasis was present in one out of every ten samples.
Expressions of Ki-67 and E-cadherin are given in  cases. 19Poorly differentiated tumors show more positive and less negative expression of Ki-67 and E-cadherin, respectively.Most of the tumors were well differentiated in this present study.This may be the probable explanation for more negative expression of Ki-67 and more positive expression of E-cadherin in this study.
He et al. 7 and Grigoras et al. 20  finding is consistent with the findings of Yang et al., 19,21 who did not find any significant correlation between Ecadherin expression with age, sex, and tumor histologic type.However, a study in Korea found a considerable correlation between the down-regulation of E-cadherin and socio-demographic characteristics like male gender and histological type 19 .He et al. 7 and Yang et al. 21found a significant association of negative expression of Ecadherin with lymph node metastasis and tumor differentiation.
Ki-67 expression may be considered a valuable marker for aggressive NSCLC and lung cancer prognosis, as suggested by a few studies. 6,12,13,22  W acknowledge some limitations of the study.We used surgically resected or core biopsy NSCLC tissue but did not get enough tissue for some of the FFPE core biopsy.
The sample employed in this study is not representative of the wider population.As seen by other studies, the sample size is also relatively small to detect real differences.

Conclusion
We observed that positive expression of Ki-67 and negative expression of E-cadherin was found in twofifths of NSCLC tissues but not significant as most of the tumors were well differentiated without distant metastasis and with minimum lymph node involvement.Simultaneous estimated of Ki-67 and Ecadherin may contribute to the treatment planning and predict prognosis of the NSCLC patients.
This cross-sectional study was done under the Department of Anatomy of the Bangabandhu Sheikh Mujib Medical University.The data were collected from January 2022 to October 2022.Initially, a total of 40 formalin-fixed paraffin-embedded (FFPE) NSCLC tissue blocks were identified based on hospital records from the Department of Pathology at the National Institute of Diseases of the Chest and Hospital and Dhaka Medical College and Hospital.Simultaneously, respective patients were communicated for further information.A total of 15 tissue samples were excluded due to insufficient tissue, prior chemotherapy or radiotherapy, and absence of patient records.Twentyfive samples were analyzed, and relevant clinicopathological data, including age, sex, and associated histopathological reports, were collected from the patients or their authorized representatives after obtaining written informed consent.Laboratory analysis was performed at the Histopathology Department of the National Institute of Cancer Research and Hospital.Identification of tumor specimens FFPE tissue blocks were cut into 4µm thick sections and incubated in a hot air oven at 60-65°C for 30 minutes.Then, clearing was done in xylene (three changes) and rehydrated gradually in decreasing concentrations (100%, 90%, 80%, 70%) of isopropyl alcohol.The slides were treated with Dako target retrieval solution in a conventional water bath and rinsed with Tris buffer solution (TBS; pH 7.4).Then, the slides were incubated in 100-150µL PRB (peroxidase blocking reagent) to block the endogenous peroxidase activity.Then the slides were incubated with primary antibodies, Ki-67 (FLEX monoclonal Mo A Hu Ki-67 Antigen, MIB-1, Dako, Denmark) and E-cadherin (FLEX monoclonal Mo A Hu E-cadherin Antigen, NCH-38, Dako, Denmark) for 30 minutes.Then, the slides were washed in TBS and incubated in horseradish peroxidase blocking reagent for 30 minutes.After that, staining and counter-staining were done with diaminobenzidine and hematoxylin, respectively.Positive controls (vermiform appendix forKi-67 and normal breast tissue for E-cadherin) were stained as described for tumor specimens.8

FIGURE 1 and FIGURE 2 ,
FIGURE 1 and FIGURE 2, respectively.Of the 25 NSCLC tissue samples, 15 exhibited positive expression for E-cadherin, while 15 were negative for Ki-67 expression.Of the 15 NSCLC tissues with positive Ecadherin expression, Ki-67 was positive in 6 cases.Conversely, among the 10 NSCLC tissues negative for Ecadherin, Ki-67 was positive in 4 cases.However, no statistically significant association (P=0.99) was

FIGURE 1 FIGURE 2
FIGURE 1 Expression of Ki-67 in non-small cell lung cancer tissue revealed an inverse correlation between the expression of E-cadherin and Ki -67 in NSCLC.These findings demonstrated that detecting lung cancer aggressiveness may benefit from potential correlations between E-cadherin and Ki-67 and assessing their level simultaneously.The present study's findings are inconsistent with these studies, possibly due to the small sample size and low percentage of poorly differentiated tumors analyzed in this study.This study found no statistically significant association between Ki-67 and E-cadherin expression with clinicopathological characteristics.A statistically significant association was found between E-cadherin expression and co-morbidity of the patients; the NSCLC patients with comorbidity (Chronic obstructive pulmonary disease/asthma) exhibited positive expression of E-cadherin in this study.On the other hand, Ki-67 expression did not show any statistically significant association with co-morbidity.No statistically significant association was found between Ki-67 and E-cadherin expression with the age, sex, histological type, tumor differentiation, lymph node metastasis, and family history of lung cancer.Our