Bovine milk casein as a cost-effective alternative blocking protein for enhancing signal-to-noise ratio in HER-2 immunohistochemistry
Article Sidebar
Main Article Content
Abstract
Background: Accurate HER-2 immunohistochemistry (IHC) assessment is essential in routine oncology diagnostics. However, the high cost of commercial blocking agents creates a financial burden for pathology laboratories, particularly in resource-limited settings.
Objectives: This study evaluated the efficacy of bovine milk casein (BMC) as a cost-effective alternative blocking protein to improve the Signal-to-Noise Ratio (SNR) while maintaining diagnostic signal intensity.
Materials and methods: A quantitative experimental approach was performed using FFPE tissue sections derived from a verified HER-2 3+ breast carcinoma cohort (N=40, 10/group). The semi-quantitative scoring stability and ImageJbased digital signal-to-noise ratio metrics were assessed by directly testing three BMC concentrations (2%, 3%, and 4%) against normal goat serum standard. Differences in ordinal scoring were assessed using non-parametric Kruskal-Wallis testing. Continuous SNR parameters were compared using One-Way ANOVA with Tukey’s post-hoc comparisons.\
Results: The 3% and 4% BMC concentrations showed 100% diagnostic consistency with a 3+ score, as the control group did. By contrast, 2% BMC significantly lowered diagnostic intensity, with all specimens (100%) reclassified as a 2+ score (Equivocal) (p<0.05, Kruskal-Wallis). Quantitative digital analysis showed that 4% BMC yielded the highest Mean SNR (6.09±0.28), statistically outperforming the control group (5.08±0.11, p<0.05).
Conclusion: Bovine milk casein at 4% concentration provides a practical and highly economical blocking alternative (>99% cost reduction) that enhances visual contrast in HER-2 IHC. While these findings support its application in resource-constrained environments, further validation across broader biomarker expression levels remains necessary to confirm widespread diagnostic stability.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Personal views expressed by the contributors in their articles are not necessarily those of the Journal of Associated Medical Sciences, Faculty of Associated Medical Sciences, Chiang Mai University.
References
Sung H, Ferlay JL, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3): 209-49. doi: 10.3322/caac.21660. DOI: https://doi.org/10.3322/caac.21660
Tan WCC, Nerurkar SN, Cai HY, Ng HHM, Wu D, Wee YTF, et al. Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun. 2020; 40(4): 135-53. doi: 10.1002/cac2.12023. DOI: https://doi.org/10.1002/cac2.12023
Kang S, Kim SB. HER2-low breast cancer: now and in the future. Cancer Res Treat. 2024; 56(3): 700-20. doi: 10.4143/crt.2023.1138. DOI: https://doi.org/10.4143/crt.2023.1138
Marchiò C, Annaratone L, Marques A, Casorzo L, Berrino E, Sapino A. Evolving concepts in HER2 evaluation in breast cancer: heterogeneity, HER2-low carcinomas and beyond. Semin Cancer Biol. 2021; 72: 123-35. doi: 10.1016/j.semcancer.2020.02.016. DOI: https://doi.org/10.1016/j.semcancer.2020.02.016
Sayed S, Cherniak W, Lawler M, Tan SY, El Sadr W, Wolf N, et al. Improving pathology and laboratory medicine in low-income and middleincome countries: roadmap to solutions. Lancet. 2018; 391(10133): 1939-52. doi: 10.1016/S0140-6736(18)30459-8. DOI: https://doi.org/10.1016/S0140-6736(18)30459-8
Ohnishi C, Ohnishi T, Ntiamoah P, Ross DS, Yamaguchi M, Yagi Y. Standardizing HER2 immunohistochemistry assessment: calibration of color and intensity variation in whole slide imaging caused by staining and scanning. Appl Microsc. 2023; 53(1): 8. doi: 10.1186/s42649-023-00091-8. DOI: https://doi.org/10.1186/s42649-023-00091-8
Kim SW, Roh J, Park CS. Immunohistochemistry for pathologists: protocols, pitfalls, and tips. J Pathol Transl Med. 2016; 50(6): 411-8. doi: 10.4132/jptm.2016.08.08. DOI: https://doi.org/10.4132/jptm.2016.08.08
Ramos-Vara JA, Borst LB. Immunohistochemistry: fundamentals and applications in oncology. In: Meuten DJ, editor. Tumors in Domestic Animals. 5th ed. Ames: John Wiley & Sons; 2017. p. 44-87. doi: 10.1002/9781119181200.ch3. DOI: https://doi.org/10.1002/9781119181200.ch3
Hoda RS, Brogi E, Wen HY. Quality issues in diagnostic immunohistochemistry in breast pathology. Pathobiology. 2022; 89(5): 324-33. doi: 10.1159/000522538. DOI: https://doi.org/10.1159/000522538
Filho OM, Viale G, Stein S, Trippa L, Yardley DA, Mayer IA, et al. Impact of HER2 heterogeneity on treatment response of early-stage HER2-positive breast cancer: phase II neoadjuvant clinical trial of T-DM1 combined with pertuzumab. Cancer Discov. 2021; 11(10): 2474-87. doi: 10.1158/2159-8290.CD-20-1557. DOI: https://doi.org/10.1158/2159-8290.CD-20-1557
Wolff AC, Somerfield MR, Dowsett M, Hammond MEH, Hayes DF, McShane LM, et al. Human epidermal growth factor receptor 2 testing in breast cancer: ASCO-College of American Pathologists guideline update. J Clin Oncol. 2023; 41(22): 3867-72. doi: 10.1200/JCO.22.02864. DOI: https://doi.org/10.1200/JCO.22.02864
Magaki S, Hojat SA, Wei B, So A, Yong WH. An introduction to the performance of immunohistochemistry. Methods Mol Biol. 2019; 1897: 289-98. doi: 10.1007/978-1-4939-8935-5_25. DOI: https://doi.org/10.1007/978-1-4939-8935-5_25
Horne DS. Casein micelle structure and stability. In: Boland M, Singh H, editors. Milk Proteins: From Expression to Food. 2nd ed. San Diego: Academic Press; 2014. p. 169-200. doi: 10.1016/B978-0-12-405171-3.00006-4. DOI: https://doi.org/10.1016/B978-0-12-405171-3.00006-4
Lossos C, Nadji M, Lossos IS, Chapman JR. Casein protein block to optimize immunohistochemistry detection of surface immunoglobulin heavy chain expression in lymphoid cells. Appl Immunohistochem Mol Morphol. 2023; 31(2): 132-4. doi: 10.1097/PAI.0000000000001091. DOI: https://doi.org/10.1097/PAI.0000000000001091
Andriola DD, Ferreira GN, Candelário JM, Piazenski IN, Biavatti M, Sakamoto LB, et al. Validated cost-effective casein-based blocking buffers for enhanced neurocysticercosis ELISA accuracy. Diagn Microbiol Infect Dis. 2025; 113(4): 117033. doi: 10.1016/j.diagmicrobio.2025.117033. DOI: https://doi.org/10.1016/j.diagmicrobio.2025.117033
Iswara A, Dewi SS, Masruro YA. HER2 immunohistochemical staining using skim milk and normal serum (in Indonesian). Biomedika. 2017; 10(1): 52-5. doi: 10.31001/biomedika.v10i1.226. DOI: https://doi.org/10.31001/biomedika.v10i1.226
Rakha EA, Chmielik E, Schmitt FC, Tan PH, Quinn CM, Gallagy G. Assessment of predictive biomarkers in breast cancer: challenges and updates. Pathobiology. 2022; 89(5): 263-77. doi: 10.1159/000525092. DOI: https://doi.org/10.1159/000525092
Stålhammar G, Fuentes Martinez N, Lippert M, Tobin NP, Mølholm I, Kis L, et al. Digital image analysis outperforms manual biomarker assessment in breast cancer. Mod Pathol. 2016; 29(4): 318-29. doi: 10.1038/modpathol.2016.34. DOI: https://doi.org/10.1038/modpathol.2016.34
Serdar CC, Cihan M, Yücel D, Serdar MA. Sample size, power and effect size revisited: simplified and practical approaches in pre-clinical, clinical and laboratory studies. Biochem Med (Zagreb). 2021; 31(1): 010502. doi: 10.11613/BM.2021.010502. DOI: https://doi.org/10.11613/BM.2021.010502
Oumarou Hama H, Aboudharam G, Barbieri R, Lepidi H, Drancourt M. Immunohistochemical diagnosis of human infectious diseases: a review. Diagn Pathol. 2022; 17(1): 17. doi: 10.1186/s13000-022-01197-5. DOI: https://doi.org/10.1186/s13000-022-01197-5
Ivanova M, Porta FM, D’Ercole M, Pescia C, Sajjadi E, Cursano G, et al. Standardized pathology report for HER2 testing in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer. Virchows Arch. 2024; 484(1): 3-14. doi: 10.1007/s00428-023-03656-w. DOI: https://doi.org/10.1007/s00428-023-03656-w
Marques SI, Carmo H, Carvalho F, Sá SI, Silva JP. A semi-automatic method for the quantification of astrocyte number and branching in bulk immunohistochemistry images. Int J Mol Sci. 2023; 24(5): 4508. doi: 10.3390/ijms24054508. DOI: https://doi.org/10.3390/ijms24054508
Sompuram SR, Vani K, Schaedle AK, Balasubramanian A, Bogen SA. Quantitative assessment of immunohistochemistry laboratory performance by measuring analytic response curves and limits of detection. Arch Pathol Lab Med. 2018; 142(7): 851-62. doi: 10.5858/arpa.2017-0330-OA. DOI: https://doi.org/10.5858/arpa.2017-0330-OA
Thanasan S, Sukhakul K, Chitpakdee S, Kitkumthorn N. Diagnostic accuracy of immunohistochemistry for HER2-positive breast cancer. Asian Pac J Cancer Prev. 2023; 24(12): 4321-7. doi: 10.31557/APJCP.2023.24.12.4321. DOI: https://doi.org/10.31557/APJCP.2023.24.12.4321
Ramos-Vara JA. Principles and methods of immunohistochemistry. Methods Mol Biol. 2017; 1641: 115-28. doi: 10.1007/978-1-4939-7172-5_5. DOI: https://doi.org/10.1007/978-1-4939-7172-5_5
Liu G, Guo B, Luo M, Sun S, Lin Q, Kan Q, et al. A comprehensive review on preparation, structureactivities relationship, and calcium bioavailability of casein phosphopeptides. Crit Rev Food Sci Nutr. 2024; 64(4): 996-1014. doi: 10.1080/10408398.2022.2111546. DOI: https://doi.org/10.1080/10408398.2022.2111546
Lara H, Li Z, Abels E, Aeffner F, Bui MM, ElGabry EA, et al. Quantitative image analysis for tissue biomarker use: a white paper from the Digital Pathology Association. Appl Immunohistochem Mol Morphol. 2021; 29(7): 479-93. doi: 10.1097/PAI.0000000000000930. DOI: https://doi.org/10.1097/PAI.0000000000000930
Rizzardi AE, Zhang X, Vogel RI, Kolb S, Geybels MS, Leung YK, et al. Quantitative comparison and reproducibility of pathologist scoring and digital image analysis of estrogen receptor β2 immunohistochemistry in prostate cancer. Diagn Pathol. 2016; 11(1): 63. doi: 10.1186/s13000-016-0511-5. DOI: https://doi.org/10.1186/s13000-016-0511-5
Mebratie DY, Dagnaw GG. Review of immunohistochemistry techniques: applications, current status, and future perspectives. Semin Diagn Pathol. 2024; 41(3): 154-60. doi: 10.1053/j.semdp.2024.05. 001. DOI: https://doi.org/10.1053/j.semdp.2024.05.001
Steiner DF, MacDonald R, Liu Y, Truszkowski P, Hipp JD, Gammage C, et al. Impact of deep learning assistance on the histopathologic review of lymph nodes for metastatic breast cancer. Am J Surg Pathol. 2018; 42(12): 1636-46. doi: 10.1097/PAS.0000000000001151. DOI: https://doi.org/10.1097/PAS.0000000000001151
Gordian-Arroyo AM, Zynger DL, Tozbikian GH. Impact of the 2018 ASCO/CAP HER2 guideline focused update. Am J Clin Pathol. 2019; 152(1): 17-26. doi: 10.1093/ajcp/aqz012. DOI: https://doi.org/10.1093/ajcp/aqz012
Dagogo-Jack I, Shaw AT. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol. 2018; 15(2): 81-94. doi: 10.1038/nrclinonc.2017.166. DOI: https://doi.org/10.1038/nrclinonc.2017.166
