Immunohistochemistry in the Study of Cancer Biomarkers for Oncology Drug Development

  • Fang Jiang
  • Evelyn M. McKeegan


Immunohistochemistry (IHC) assays can identify tumor biomarkers and provide valuable information for oncology drug development. This chapter describes the basic IHC assay step by step to identify target proteins and assess protein expression levels and subcellular localization. The steps are tissue preparation, fixation, selection of antibody and controls, detection system, counterstain, coverslip, score, and troubleshooting. The chapter also discusses the use of IHC assays in cancer biomarker studies, in particular the application of a multi-fluorophore labeling technique to study tumor vasculature in preclinical human xenograft tumor models for antiangiogenic drug development, and the use of an immunocytochemistry (ICC) assay on circulating tumor cell isolated from clinical samples. The study data demonstrate that IHC assays provide important information for cancer biomarker studies, illuminating the mechanisms of action for oncology drugs. IHC arrays are indispensable tools in histogenic assessment and biomarker development.


Immunohistochemistry Angiogenesis Tumor vascular measurement Circulating tumor cell Antigen retrieval Cancer biomarker 



The authors thank Dr. Neela X. Patel for thoughtful suggestions and editing on this chapter.


  1. 1.
    Elias JM. Immunohistochemical methods. In: Elias JM, editor. Immunohistopathology. A practical approach to diagnosis. 2nd ed. Chicago, IL: ASCP; 2003.Google Scholar
  2. 2.
    Lynch F, Bernstein S. Immunohistochemistry assays in drug development performed by a contract research laboratory. In: Platero JS, editor. Molecular pathology in drug discovery and development. Hoboken, NJ: Wiley; 2009.Google Scholar
  3. 3.
    Ramos-Vara JA. Technical aspects of immunohistochemistry. Vet Pathol. 2005;42:405–26.CrossRefPubMedGoogle Scholar
  4. 4.
    Wilson C, Schulz S, Waldman SA. Biomarker development, commercialization, and regulation: individualization of medicine lost in translation. Clin Pharm Ther. 2007;2:153–5.CrossRefGoogle Scholar
  5. 5.
    Bartlett JMS, Going JJ, Mallon EA, Watters AD, Reeves JR, Stanton P, Richmond J, Donald B, Ferrier R, Cooke TG. Evaluating HER2 amplification and overexpression in breast cancer. J Pathol. 2001;195:422–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Pantel K, Brakenhoff RH, Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nat Rev Can. 2008. doi: 10.1038/nrc2375.Google Scholar
  7. 7.
    Santos GdC, Shepherd FA, Tsao MS. EGFR mutation and lung cancer: mechanisms of disease. Annu Rev Pathol. 2011;6:49–69.Google Scholar
  8. 8.
    Samoszuk MK, Walter J, Mechetner E. Improved immunohistochemical method for detecting hypoxia gradients in mouse tissues and tumors. J Histochem Cytochem. 2004;52(6):837–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Jain RK, Duda DG, Willett CG, Sahani DV, Zhu AX, Loeffler JS, Batchelor T, Sorensen G. Biomarkers of response and resistance to antiangiogenic therapy. Nat Rev Clin Oncol. 2009;6:327–38.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Li JL, Harris AL. The potential of new tumor endothelium-specific markers for the development of antivascular therapy. Cancer Cell. 2007;11:478–81.CrossRefPubMedGoogle Scholar
  11. 11.
    Seaman S, Stevens J, Yang MY, Logsdon D, Graff-Cherry C, St. Croix B. Genes that distinguish physiological and pathological angiogenesis. Cancer Cell. 2007;11:539–54.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sharma RA, Harris AL, Galgleish AG, Steward WP, O’Byrne KJ. Angiogenesis as a biomarker and target in cancer chemoprevention. Lancet Oncol. 2001;2:726–32.CrossRefPubMedGoogle Scholar
  13. 13.
    Faccioli N, Marzola P, Boschi F, Sbarbati A, D’Onofrio M, Mucelli RP. Pathological animal models in the experimental evaluation of tumour microvasculature with magnetic resonance imaging. Radiol Med. 2007;112:319–28.CrossRefPubMedGoogle Scholar
  14. 14.
    Sikora J, Dworacki G, Trybus M, Batura-Gabryel H, Zeromski J. Correlation between DNA content, expression of Ki-67 antigen of tumor cells and immunophenotype of lymphocytes from malignant pleural effusions. Tumor Biol. 1998;19:196–204.CrossRefGoogle Scholar
  15. 15.
    Trusheim MR, Berndt ER, Douglas FL. Stratified medicine: strategic and economic implications of combining drugs and clinical biomarkers. Nat Rev. 2007;6:287–93.Google Scholar
  16. 16.
    Batchelor TT, Sorensen AG, Tomaso ED, Zhang WT, Duda DG, Cohen KS, Kozak KR, Cahill DP, Chen PJ, Zhu MW, Ancukiewicz M, Mrugala MM, Plotkin S, Drappatz J, Louis DN, Ivy P, Scadden DT, Benner T, Loeffler JS, Wen PY, Jain RK. AZD2171, a Pan-VEGF receptor tyrosine kinas inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer Cell. 2007;11:83–95.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Brustmann H, Riss P, Naude S. The relevance of angiogenesis in benign and malignant epithelial tumor s of the ovary: a quantitative histological study. Gynecol Oncol. 1997;67:20–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Chiba Y, Taniquchi T, Matsuyama K, Sasaki M, Kato Y, Tanaka H, Muraoka R, Tanigawa N. Tumor angiogenesis, apoptosis and p53 oncogene in stage I lung adenocarcinoma. Surg Today. 1999;29:1148–53.CrossRefPubMedGoogle Scholar
  19. 19.
    Fox SB, Leek RD, Weekes MP, Whitehouse RM, Gatter KC, Harris AL. Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. J Pathol. 1995;177:275–83.CrossRefPubMedGoogle Scholar
  20. 20.
    Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol. 1993;143:401–9.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniem OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4:844–7.CrossRefPubMedGoogle Scholar
  22. 22.
    Wan WH, Fortuna MB, Furmanski P. A rapid and efficient method for testing immunohistochemical reactivity of monoclonal antibodies against multiple tissue samples simultaneously. J Immunol Methods. 1987;103:121–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Hayat MA. Fixation and embedding. In: Hayat MA, editor. Microscopy, immunohistochemistry, and antigen retrieval methods for light and electron microscopy. New York: Kluwer Academic; 2002.Google Scholar
  24. 24.
    Dapson RW. Fixation for the 1990’s: a review of needs and accomplishments. Biotech Histochem. 1993;68:75–82.CrossRefPubMedGoogle Scholar
  25. 25.
    Jiang F, Albert DH, Luo YP, Tapang P, Zhang K, Davidsen SK, Fox GB, Lesniewski R, McKeegan EM. ABT-869, a multi-targeted receptor tyrosine kinase inhibitor, reduces tumor microvascularity and improves vascular wall integrity in preclinical tumor models. JPET. 2011;338:134–42.CrossRefGoogle Scholar
  26. 26.
    Luo Y, Jiang F, Cole TB, Hradil VP, Reuter D, Chakravartty A, Albert DH, Davidsen SK, Cox BF, McKeegan EM, Fox GB. A novel multi-targeted tyrosine kinase inhibitor, linifanib (ABT-869), produces functional and structural changes in tumor vasculature in an orthotopic rat glioma model. Cancer Chemother Pharmacol. 2012;69(4):911–21 [Epub ahead of print], PMID:22080168 [PubMed—as supplied by publisher].CrossRefPubMedGoogle Scholar
  27. 27.
    Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory and practice of fixation of tissues. J Histotechnol. 2001;24:173–90.CrossRefGoogle Scholar
  28. 28.
    Shi SR, Cote RJ, Taylor CR. Antigen retrieval techniques: current perspectives. J Histochem Cytochem. 2001;49:931–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Riera JR, Atengo-Osuna C, Longmate JA, Battifora H. The immunohistochemical diagnostic panel for epithelial mesothelioma: a reevaluation after heat-induced epitope retrieval. Am J Surg Pathol. 1997;12:1395–8.Google Scholar
  30. 30.
    Burry RW. Specificity controls for immunocytochemical methods. J Histochem Cytochem. 2000;48(2):163–5.CrossRefPubMedGoogle Scholar
  31. 31.
    Albert DH, Tapang P, Magoc TJ, Pease LJ, Reuter DR, Wei RQ, Li J, Guo J, Bousquet PF, Ghoreishi-Haack NS, Wang B, Bukofzer GT, Wang YC, Stavropoulos JA, Hartandi K, Niquette AL, Soni N, Johnson EF, McCall JO, Bouska JJ, Luo Y, Donawho CK, Dai Y, Marcotte PA, Glaser KB, Michaelides MR, Davidsen SK. Preclinical activity of ABT-869, a multi targeted receptor tyrosine kinase inhibitor. Mol Cancer Ther. 2006;5(4):995–1006.CrossRefPubMedGoogle Scholar
  32. 32.
    Taylor CR, Shi S-R, Barr NJ, Wu N. Techniques of immunohistochemistry: principles, pitfalls, and standardization. In: Dabbs DJ, editor. Diagnostic immunohistochemistry. New York: Churchill Livingstone; 2002.Google Scholar
  33. 33.
    Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29:577–80.CrossRefPubMedGoogle Scholar
  34. 34.
    Xu J, Stolk JA, Zhang X, Silva SJ, Houghton RL, Matsumura M, Vedivick TS, Leslie KB, Badaro R, Reed SG. Identification of differentially expressed genes in human prostate cancer using subtraction and microarray. Cancer Res. 2000;60:1677–82.PubMedGoogle Scholar
  35. 35.
    Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LWMM, Hayes DF. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004;351:781–91.CrossRefPubMedGoogle Scholar
  36. 36.
    Giordano A, Giuliano M, De Laurentiis M, Arpino G, Jackson S, Handy BC, Ueno NT, Andreopoulou E, Alvarez RH, Valero V, De Placido S, Hortobagyi GN, Reuben JM, Cristofanilli M. Circulating tumor cells in immunohistochemical subtypes of metastatic breast cancer: lack of prediction in HER2-positive disease treated with targeted therapy. Ann Oncol. 2012;23(5):1144–50 [Epub ahead of print].CrossRefPubMedGoogle Scholar
  37. 37.
    Gandhi L, Camidge DR, Ribeiro de Oliveira M, Bonomi P, Gandara D, Khaira D, Hann CL, McKeegan EM, Litvinovich E, Hemken PM, Dive C, Enschede SH, Nolan C, Chiu YL, Busman T, Xiong H, Krivoshik AP, Humerickhouse R, Shapiro GI, Rudin CM. A phase 1 study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in subjects with small cell lung cancer (SCLC) and other solid tumors. J Clin Oncol. 2011;29(7):909–16.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Bauer KD, Torre-Bueno JDL, Diel IJ, Hawes D, Decker WJ, Priddy C, Bossy B, Ludmann S, Yamamote K, Masih AS, Espinoza FP, Harrington DS. Reliable and sensitive analysis of occult bone marrow metastases using automated cellular imaging. Clin Can Res. 2000;3552(6):3552–9.Google Scholar
  39. 39.
    McCabe A, Dolled-Filhart D, Camp RL, Rimm DL. Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis. J Natl Cancer Inst. 2005;97(24):1808–15.CrossRefPubMedGoogle Scholar
  40. 40.
    Debbage PL, Griebel J, Ried M, Gneiting T, DeVries A, Hutzler P. Lectin intravital perfusion studies in tumor-bearing mice: micrometer-resolution, wide area mapping of microvascular labeling, distinguishing efficiently and inefficiently perfused microregions in the tumor. J Histochem Cytochem. 1998;46:627–39.CrossRefPubMedGoogle Scholar
  41. 41.
    Gee MS, Procopio WN, Makonnen S, Feldman MD, Yeilding NM, Lee WMF. Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy. Am J Pathol. 2003;162:183–93.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G, Roberge S, Jain RK, McDonald DM. Openings between detective endothelial cells explain tumor vessel leakiness. Am J Pathol. 2000;156:363–80.CrossRefGoogle Scholar
  43. 43.
    Minamikawa T, Miyake T, Takamatsu T, Fujita S. A new method of lectin histochemistry for the study of brain angiogenesis. Histochemistry. 1987;87:317–20.CrossRefPubMedGoogle Scholar
  44. 44.
    Morikawa S, Baluk P, Kaidoh T, Haskell A, Jain RK, McDonald DM. Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors. Am J Pathol. 2002;160:985–1000.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Dings RPM, Loren M, Heun H, McNeil E, Griffioen AW, Mayo KH, Griffin RJ. Scheduling of radiation with angiogenesis inhibitors anginex and avastin improves therapeutic outcome via vessel normalization. Clin Cancer Res. 2007;13:3395–402.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Raleigh JA, Chou SC, Arteel GE, Horsman MR. Comparisons among pimonidazole binding, oxygen electrode measurements, and radiation response in C3H mouse tumors. Radiat Res. 1999;151:580–9.CrossRefPubMedGoogle Scholar
  47. 47.
    Raleigh JA, Chou SC, Calkins-Adams DP, Ballenger CA, Novotny DB, Varia MA. A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas. Clin Cancer Res. 2000;6:855–62.PubMedGoogle Scholar
  48. 48.
    Varia MA, Calkins-Adams DP, Rinker LH, Kennedy AS, Novotny DB, Fowler Jr WC, Raleigh JA. Pimonidazole: a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma. Gynecol Oncol. 1998;71:270–7.CrossRefPubMedGoogle Scholar
  49. 49.
    Middleton M, Friedlander P, Hamid O, Daud A, Plummer R, Falotico N, Chyla B, Jiang F, McKeegan E, Mostafa NM, Zhu M, Qian J, McKee M, Luo Y, Giranda VL, McArthur GA. Randomized phase II study evaluating veliparib (ABT-888) with temozolomide in patients with metastatic melanoma. Ann Oncol. 2015;26:2173–9.Google Scholar
  50. 50.
    Camp RL, Chung GG, Rimm DL. Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med. 2002;8:1323–7.CrossRefPubMedGoogle Scholar
  51. 51.
    Food and Drug Administration. Medical device: guidance for submission of immunohistochemistry applications to the FDA; final guidance for industry. 2008.
  52. 52.
    Mass R. The role of HER-2 expression in predicting response to therapy in breast cancer. Semin Oncol. 2000;27:46–52.PubMedGoogle Scholar
  53. 53.
    Ravdin PM. Should HER2 status be routinely measured for all breast cancer patients? Semin Oncol. 1999;26:117–23.PubMedGoogle Scholar
  54. 54.
    Bertheau P, Cazals-Hatem D, Meignnin V, de Roquancourt A, Verola O, Lesourd A, Sene C, Brocheriou C, Janin A. Variability of immunohistochemical reactivity on stored paraffin slides. J Clin Pathol. 1998;51:370–4.CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Jacobs TW, Prioleau JE, Stillman IE, Schnitt SJ. Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst. 1996;88:1054–9.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Cancer Discovery, Global Pharmaceutical Research and DevelopmentAbbott LaboratoriesAbbott ParkUSA

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