Document Type : Original Article
Authors
- Masoud Hassanzadeh Makoui 1
- Maryam Mobini 1
- Jalal Khoshnoodi 1
- Tannaz Bahadori 1
- Forough Golsaz-Shirazi 1
- Hedieh Moradi Tabriz 2
- Zahra Madjd 3
- Mahmood Jeddi-Tehrani 4
- Amir-Hassan Zarnani 1
- Mohammad Mehdi Amiri 1
- Fazel Shokri 1
1 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Pathology, Iran University of Medical Sciences, Tehran, Iran
4 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Abstract
Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test.
Objective: To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes.
Methods: Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs.
Results: Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer.
Conclusion: The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.
Keywords
- Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2018;68(6): 394-424.
- Goossens N, Nakagawa S, Sun X, Hoshida Y. Cancer biomarker discovery and validation. Transl Cancer Res. 2015;4(3): 256-269.
- Zahavi D, Weiner L. Monoclonal Antibodies in Cancer Therapy. Antibodies (Basel). 2020;9(3): 34-54.
- Miaomiao T, Shu C, Xianquan Z, Qi Z. Ki-67 labeling index is a predictive marker for a pathological complete response to neoadjuvant chemotherapy in breast cancer: A meta-analysis. Medicine. 2017; 96(51): e9384.
- Chen MB, Wu XY, Yu R, Li C, Wang LQ, Shen W, et al. P53 status as a predictive biomarker for patients receiving neoadjuvant radiation-based treatment: a meta-analysis in rectal cancer. PLoS One. 2012;7(9):e45388.
- Mehta S, Shelling A, Muthukaruppan A, Lasham A, Blenkiron C, Laking G, et al. Predictive and prognostic molecular markers for cancer medicine. Ther Adv Med Oncol. 2010;2(2): 125-148.
- Personalized medicine: identifying the appropriate patient through biomarkers in oncology. P T. 2011; 36(7 Suppl 1): 3-10.
- Mansouri H, Mnango LF, Magorosa EP, Sauli E, Mpolya EA. Ki-67, p53 and BCL-2 Expressions and their Association with Clinical Histopathology of Breast Cancer among Women in Tanzania. Sci Rep. 2019;9: 9918.
- DeLeo AB, Jay G, Appella E, Dubois GC, Law LW, Old LJ. Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse. Proc Natl Acad Sci U S A. 1979;76(5):2420-2424.
- Fridman J, Lowe S. Control of apoptosis by p53. Oncogene. 2003;22: 9030–9040.
- Ozaki T, Nakagawara A. Role of p53 in Cell Death and Human Cancers. Cancers (Basel). 2011;3(1): 994-1013.
- Yang P, Du CW, Kwan M, Liang SX, Zhang GJ. The impact of p53 in predicting clinical outcome of breast cancer patients with visceral metastasis. Sci Rep. 2013;3: 2246.
- Sobhani N, D'Angelo A, Wang X, Young KH, Generali D, Li Y. Mutant p53 as an Antigen in Cancer Immunotherapy. Int J Mol Sci. 2020;21(11): 4087.
- Fasanella S, Leonardi E, Cantaloni C, Eccher C, Bazzanella I, Aldovini D, et al. Proliferative activity in human breast cancer: Ki-67 automated evaluation and the influence of different Ki-67 equivalent antibodies. Diagn Pathol. 2011;6(S7): 1-6.
- Peng H, Tan X, Wang Y, Dai L, Liang G, Guo J, et al. Clinical significance of Ki67 and circulating tumor cells with an epithelial-mesenchymal transition phenotype in non-small cell lung cancer. Am J Transl Res. 2020;12(6): 2916-2928.
- Soliman NA, Yussif SM. Ki-67 as a prognostic marker according to breast cancer molecular subtype. Cancer Biol Med. 2016;13(4):496-504.
- Chen WJ, He DS, Tang RX, Ren FH, Chen G. Ki-67 is a valuable prognostic factor in gliomas: evidence from a systematic review and meta-analysis. Asian Pac J Cancer Prev. 2015;16(2):411-420.
- Takkem A, Barakat C, Zakaraia S, Zaid K, Najmeh J, Ayoub M, et al. Ki-67 Prognostic Value in Different Histological Grades of Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma. Asian Pac J Cancer Prev. 2018;19(11):3279-3286.
- Fasching PA, Heusinger K, Haeberle L, Niklos M, Hein A, Bayer CM, et al. Ki67, chemotherapy response, and prognosis in breast cancer patients receiving neoadjuvant treatment. BMC Cancer. 2011;11: 486.
- Melling N, Kowitz CM, Simon R, Bokemeyer C, Terracciano L, Sauter G, et al. High Ki67 expression is an independent good prognostic marker in colorectal cancer. Journal of Clinical Pathology. 2016;69: 209-214.
- Matos LL, Trufelli DC, de Matos MG, da Silva Pinhal MA. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark Insights. 2010;5: 9-20.
- Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer. 1983;31: 13-20.
- Legros Y, Lacabanne V, d'Agay MF, Larsen CJ, Pla M, Soussi T. Production of human p53 specific monoclonal antibodies and their use in immunohistochemical studies of tumor cells. Bulletin du Cancer. 1993;80(2): 102-110.
- Mahmoudi AR, Ghods R, Madjd Z, Abolhasani M, Saeednejad Zanjani L, Safaei M, et al. Expression profiling of RTL1 in human breast cancer tissues and cell lines. Exp Mol Pathol. 2021;121: 104654.
- Zarei O, Irajian GR, Zarnani AH, Chamani-Tabriz L, Emami S, Jeddi-Tehrani M, et al. Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium. Avicenna J Med Biotechnol. 2011;3(2):79-85.
- Hassanzadeh Makoui M, Mobini M, Khoshnoodi J, Judaki MA, Bahadori T, Ahmadi Zare H, et al. Generation and Characterization of Novel Diagnostic Mouse Monoclonal Antibodies Against Human Estrogen Receptor Alpha and Progesterone Receptor. Asian Pac J Cancer Prev. 2022; 23(9): 2999-3007.
- Robertson S, Acs B, Lippert M, Hartman J. Prognostic potential of automated Ki67 evaluation in breast cancer: different hot spot definitions versus true global score. Breast Cancer Res Treat. 2020;183(1):161-75.
- Kikuchi S, Nishimura R, Osako T, Okumura Y, Nishiyama Y, Toyozumi Y, et al. Definition of p53 Overexpression and its Association with the Clinicopathological Features in Luminal/HER2-negative Breast Cancer. Anticancer Research. 2013;33(9):3891-7.
- MedCalc Software Ltd. Diagnostic test evaluation calculator. https://www.medcalc.org/calc/diagnostic_test.php (Version 20.113; accessed July 31, 2022).
- Kazemi T, Tahmasebi F, Bayat AA, Mohajer N, Khoshnoodi J, Jeddi-Tehrani M, et al. Characterization of novel murine monoclonal antibodies directed against the extracellular domain of human HER2 tyrosine kinase receptor. Hybridoma. 2011;30(4): 347-353.
- Li LT, Jiang G, Chen Q, Zheng JN."Ki67 is a promising molecular target in the diagnosis of cancer (Review)". Molecular Medicine Reports. 2015;11(3): 1566-1572.
- Malamou-Mitsi V, Gogas H, Dafni U, Bourli A, Fillipidis T, Sotiropoulou M. Evaluation of the prognostic and predictive value of P53 and Bcl-2 in breast cancer patiets participating in a randomized study with dose-dense sequential adjuvant chemotherapy. Annals of Oncology. 2006;17(10): 1504-1511.
- Lindboe CF, Torp SH. Comparison of Ki-67 equivalent antibodies. J Clin Pathol. 2002;55(6): 467-471.
- Uxa S, Castillo-Binder P, Kohler R, Stangner K, Muller GA, Engeland K. Ki-67 gene expression. Cell Death Differ.2021;28: 3357–3370.
- Owens R, Gilmore E, Bingham V, Cardwell C, McBride H, McQuaid S, et al. Comparison of different anti-Ki67 antibody clones and hot-spot sizes for assessing proliferative index and grading in pancreatic neuroendocrine tumours using manual and image analysis. Histopathology. 2020;77(4): 646-658.
- Turpeinen M, Serpi R, Rahkolin M, Vähäkangas K. Comparison of anti-p53 antibodies in immunoblotting. Biochem Biophys Res Commun. 2002;293(2): 850-856.
- Bonsing BA, Corver WE, Gorsira MCB, Vliet MV, Oud PS, Cornelisse CJ, et al. Specificity of seven monoclonal antibodies against p53 evaluated with Western blotting, immunohistochemistry, confocal laser scanning microscopy, and flow cytometry. Cytometry. 1997;28(1): 11-24.
- Sp N, Kang DY, Lee JM, Bae SW, Jang KJ. Potential Antitumor Effects of 6-Gingerol in p53-Dependent Mitochondrial Apoptosis and Inhibition of Tumor Sphere Formation in Breast Cancer Cells. Int J Mol Sci. 2021;22(9): 4660.
- Heaster TM, Walsh AJ, Zhao Y, Hiebert SW, Skala MC. Autofluorescence imaging identifies tumor cell-cycle status on a single-cell level. J Biophotonics. 2018;11(1): 1-26.
- Brotherick I, Shenton BK, Cowan WK, Angus B, Horne CH, Higgs MJ, Lennard TW. P53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer. Cancer Immunol Immunother. 1995;41(3): 146-150.
- Yin Y, Zeng K, Wu M, Ding Y, Zhao M, Chen Q. The levels of Ki-67 positive are positively associated with lymph node metastasis in invasive ductal breast cancer. Cell Biochem Biophys. 2014;70(2):1145-1151.
- Grabowski JP, Glajzer J, Richter R, Plett H, Muallem MZ, Braicu EI, et al. Lymphovascular space invasion and Ki67 as predictors of lymph node metastasis in primary low grade serous ovarian cancer. Int J Gynecol Cancer. 2021;31(1):98-103.
- Bostanci ME, Mollaoglu MC, Karadayi K. The relationship between Ki-67 index and axillary lymph node metastasis in breast carcinoma. Ann Med Res. 2020; 27(7):1956-1960.
- Allred DC, Clark GM, Elledge R, Fuqua SA, Brown RW, Chamness GC, et al. Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer. J Natl Cancer Inst. 1993;85(3):200-206.
)